Murata Kazuyoshi, Kaneko Yasuko
National Institute for Physiological Sciences;
Graduate School of Science and Engineering, Saitama University.
J Vis Exp. 2018 Jul 17(137):57197. doi: 10.3791/57197.
This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to occur in some cyanobacteria before cell division. However, due to the large cell size and the transient character, it is difficult to investigate the structure in detail. To overcome the difficulties, first, DNA compaction is reproducibly produced in the cyanobacterium Synechococcus elongatus PCC 7942 by synchronous culture using 12 h each light/dark cycle. Second, DNA compaction is monitored by fluorescence microscopy and captured by rapid freezing. Third, the detailed structure of DNA compacted cells is visualized in three dimensions (3D) by high voltage cryo-electron tomography. This set of methods is widely applicable to investigate transient structures in bacteria, e.g. cell division, chromosome segregation, phage infection etc., which are monitored by fluorescence microscopy and directly visualized by cryo-electron tomography at appropriate time points.
本方案描述了如何在蓝细菌中可视化瞬时DNA压缩。DNA压缩是一种最近发现的在某些蓝细菌细胞分裂前发生的显著的细胞质事件。然而，由于细胞体积大且具有瞬时性，很难对其结构进行详细研究。为了克服这些困难，首先，通过使用12小时光照/黑暗周期的同步培养，在聚球藻属细长聚球藻PCC 7942蓝细菌中可重复地产生DNA压缩。其次，通过荧光显微镜监测DNA压缩，并通过快速冷冻进行捕获。第三，通过高压冷冻电子断层扫描在三维（3D）中可视化DNA压缩细胞的详细结构。这组方法广泛适用于研究细菌中的瞬时结构，例如细胞分裂、染色体分离、噬菌体感染等，这些结构通过荧光显微镜进行监测，并在适当的时间点通过冷冻电子断层扫描直接可视化。
